I am using a fairly standard PCR protocol to produce DNA which I am then
checking by running the reaction product on a 0.5X TBE 2% agarose gel,
identifying the band of interest, then cutting this out and extracting the
DNA with the GeneClean kit. I have two problems.
First, before running on the gel, I reduce the volume of the PCR reaction
product from 40 microliters by adding NaOAC to 300mM, 2 volumes of EtOH,
precipitating and spinning, then resuspending the "pellet" in 10
microliters of water to which I add 2 microliters of "Manniatis type II"
loading buffer. However, many times this has produced a gel which looks as
if much of the DNA has remained in the well and with considerable smearing
rather than nice discreet bands. Sometimes though, it does work OK.
The second problem is that a further round of reamplification on the
GeneCleaned product has never produced decent results. I can't understand
why this should be.
Any advice on these two problems would be greatly appreciated.
--
Mike Joiner
Radiation Oncology
University of Pennsylvania joiner at pobox.upenn.edu